The response of endocrine-regulated cells depends on both the binding and degradation of regulatory hormones. although the degradation of insulin has been extensively studied with whole animals, perfused organs, suspensions of cells and purified enzymes, it is still not clear how insulin is degraded. One enzyme present in the microsomes of many cells, glutathione-insulin transhydrogenase (GIT), splits insulin into its A and B chains. Another enzyme present in the cytosol of many cells, insulin degrading enzyme (IDE), cleaves several peptide bonds in the insulin backbone. However, before insulin can be degraded by either of these enzymes it must first be bound by another molecule, the insulin receptor, and taken up by the cell. In the proposed research, the functional, structural and genetic interactions of these three molecules will be studied. First, the insulin degrading enzyme will be purified from a cell line of human lymphocytes. This purified enzyme will be characterized and both monoclonal and polyclonal antibodies will be produced against it. Second, mixtures of the purified IDE, GIT and insulin receptor will be tested for their ability to degrade insulin. In addition, the possibility that the structure of the insulin receptor is effected by these enzymes will be examined. Third, the structural linkage of plasma membrane IDE and the insulin receptor will be studied by capping each molecule on the surface of the cell and determining whether the other molecule is present in the same cap. And finally, a collection of hybrid cells will be constructed containing one or several human chronosomes on the background of a mouse fibroblast cell line. The presence of human IDE, GIT and the insulin receptor in these cells will be correlated with the presence of various human chromosomes to determine which chromosomes contain the genes encoding for these molecules.